作者感谢Leif Oxburgh和Derek Adams慷慨分享他们的知识，Leif Oxburgh对手稿有帮助的评论，StefanWölfl和UlrikeMüller的技术支持和Saskia Schmitteckert，Julia Gobbert，Sascha Weyer和Viola Mayer在实验室。这项工作得到了"生物学家公司" （CP）发展部门的支持。

根据瑞典法规和欧盟立法（2010/63 / EU）维护小鼠。所有程序均按照瑞典伦理委员会的准则进行（许可证C79 / 9，C248 / 11和C135 / 14）。海德堡大学涉及动物科目的程序已获得卡尔斯鲁厄大学和海德堡大学动物福利干事的批准。 1.培养试剂和材料的制备注意:使用层流罩以尽量减少污染。 <ol><li>在解剖当天，通过在无菌磷酸盐缓冲盐水（PBS）中将其与抗人Fc山羊抗体以1:5的摩尔比混合，将人Fc单独或重组嵌合ephrin蛋白融合至人Fc。在37℃孵育1小时5 。 </li><li>通过补充Dulbecco&#39;s Modified Eagle Medium:Nutrient来制备培养基混合物F-12（DMEM / F12）与1％青霉素/链霉素（v / v），1％谷氨酰胺（v / v），1％胰岛素转铁蛋白硒（v / v），50μg/转铁蛋白和1.5μg/ mL两性霉素。 注意:10毫升的培养基足以用于16个胚胎。 </li><li>将最终浓度为20μg/ mL的聚簇重组ephrin溶液加入到培养基中。使用聚簇人Fc作为对照。 </li><li>通过向无菌，未涂覆的平底聚苯乙烯4孔板的每个孔中加入500μL培养基来制备培养板。 </li><li>使用无菌镊子，小心地将一个孔径为5μm的聚碳酸酯膜过滤器放在介质顶部。将准备的板转移到培养箱（37℃/ 5％CO 2 ）。确保过滤器在上表面保持干燥并漂浮。 注意:一个过滤器足够用于两个肾脏成像。 </li><li>使用剃刀刀片切割大约30个吸头（体积:100μL）近似值距离尖端1 mm，导致更宽的开口。将提示放回移液管尖架。 </li></ol> 2. E11.5处的Methephric Rudiments的解剖<ol><li>通过颈椎脱位（或使用当地道德委员会批准的方法），牺牲E11.5的定时怀孕小鼠。 </li><li>去除子宫和胚胎，如Brown 等人 6所述 。从这一点开始，在夹层显微镜下工作。 </li><li>使用5号制表钳，将胚胎直接切割在前肢下方。每个胚胎使用新鲜的陪替氏培养皿。去除胚胎的腿和尾巴。为了做到这一点，用一对5号制表钳抓住要去除的组织，并使用第二对的尖端切割。 </li><li> 为了去除腹侧器官体积，使用一对镊子的尖端以朝向头尾方向横向打开胚胎体壁。 <ol><li>表面切割，平行于t他背侧主动脉。使用充血的，因此可见的背侧主动脉取向。使用封闭镊子的尖端小心地将体壁的腹部与背部分开，并将胚胎翻转过来。如前面那样，以朝向头尾方向横向切割体壁，并使用背侧主动脉取向。 </li></ol></li><li>一旦腹侧体壁已经脱离背部，用一对镊子抓住它，并仔细拉扯与器官块一起去除它。 注意:甲肾上腺素通常留在背部的身体壁上。它们是椭圆形，约200μm长的结构位于后肢的水平。 </li><li>用一对镊子握住背部侧壁，腹侧朝上。使用另一对镊子，在其末端抓住背侧主动脉，并将背侧主动脉从背侧皮瓣小心地剥离。 注意:肾脏肾脏通常保留背侧主动脉。 </li><li>使用一对镊子，切割到肾脏，以除去主动脉。然后，使用镊子的尖端仔细分离两个肾脏成胶质细胞。 </li><li>使用镊子的尖端小心地从肾脏中去除不需要的组织。注意不要损伤间质，因为损伤可能导致不良生长和排除外植体进一步分析。 </li><li> 使用微量移液管和10μLPBS中的大开口移液管头将肾脏成分分别转移到4孔板中的聚碳酸酯膜过滤器上。 注意:由于表面张力，外植体将被薄层介质覆盖。一个过滤器对于两个肾脏成像是足够的。 <ol><li>将两个肾脏中的每一个放置在过滤器的每个相应一半的中心。将板转移到37℃/ 5％CO 2的培养箱中。将板放在孵化器中，直到metanephric rud来自下一个胚胎的图像准备放在过滤器上。 </li></ol></li><li>对每个胚胎重复步骤2.3-2.9.1。 注意:通过一些实践，每个胚胎的解剖程序将需要约5分钟。 </li><li>在37℃/ 5％CO 2下孵育肾脏成像3天。 </li></ol> 3.固定和染色试剂的制备<ol><li>为了在没有钙和镁（w / v）的PBS中制备4％多聚甲醛（PFA），将PFA在60℃下在PBS中溶解，继续搅拌。冷却至室温，并使用纸过滤器去除剩余的颗粒。等分并保持在4°C即时使用或冻结长期储存。 小心:PFA有毒。戴上本地安全标准中规定的个人防护装备，并在通风橱中工作。根据制度准则废弃废物，并使用指定的废物容器。 </li><li>为了制备透化溶液，稀释Triton X-100在不含钙和镁的PBS中至0.3％（v / v）。 </li><li>为了制备封闭溶液，将不含钙和镁的5％（v / v）山羊血清和0.1％Triton X-100加入PBS中。加入叠氮化钠至终浓度为0.02％（w / v）。储存于4°C。 小心:叠氮化钠是有毒的。按照当地的安全环保规定处理叠氮化钠。 注意:使用抗体进行染色时，请使用来自二抗的物种的5％血清以制备阻断溶液。 </li><li>稀释生物素化的双歧杆菌凝集素1:200在封闭溶液中。稀释Alexa488缀合的链霉抗生物素蛋白1:200在封闭溶液中。 </li><li> 为了制备即用型嵌入介质（参见材料表），向0.1M Tris-HCl（pH8.5）中的25％（w / v）甘油中加入0.1g水溶性聚乙烯醇粘膜粘附剂/ mL， 。在连续搅拌下加热至50℃1小时，冷却，并使用1调节pH至8.0-8.5M NaOH。避免较高的NaOH浓度。加入100μg/ mL的N-丙酸镓聚糖和硫柳汞，最终浓度为0.02％（w / v）。在室温下搅拌30分钟。 小心:硫柳汞是有毒的。按照当地的安全环保法规处理。 <ol><li>将包埋介质填充在50mL管中，平衡转子，并以3,200 xg和室温离心10分钟。将澄清溶液倒入15 mL管中，弃去沉淀。等分并在-20°C储存数周。解冻后，将溶液保持在4°C。使用前立即温至〜30°C。 </li></ol></li><li>准备尽可能多的玻片，作为带有外植体的过滤器。为此，使用即时粘合剂在玻璃滑块的两侧上粘贴18 x 18毫米的两个小盖玻片，在其间留出约15毫米的过滤器空间。 </li></ol> 4.固定和染色<ol><li>培养期3次天体外 （div），从培养箱中取出板，并使用微量移液管小心地从每个孔抽取培养基。确保不要接触过滤器，并保持顶部开口朝向井壁。 </li><li>向每个孔中加入500μL的4％PFA / PBS。过滤器将浮在固定剂上。使用微量移液器，小心地滴加PFA固定剂以淹没过滤器。在室温下孵育30分钟。 注意:对于所有以下步骤，必须注意不要从过滤器上清除外植体。将吸头的开口保持在井的墙壁上。 </li><li>取出PFA，用500μLPBS冲洗两次，浸没过滤器。向每个孔中加入500μL透化溶液，并在室温下孵育1小时。 </li><li>用500μLPBS冲洗两次，并向每个孔中加入500μL封闭溶液。在室温下孵育1小时。 </li><li>用更换阻塞溶液将250μL生物素化的双歧杆菌凝集素在封闭溶液中以1:200稀释，并在4℃下孵育24小时。 </li><li>取出Dolichorus biflorus凝集素溶液，每孔用500μLPBS冲洗两次。 </li><li>在封闭溶液中加入250μL以1:200稀释的Alexa488缀合的链霉抗生物素蛋白，并在4℃下孵育24小时。或者，在室温下孵育2小时。 注意:当在4℃下孵育时，获得更好的信噪比。 </li><li>每孔用500μLPBS冲洗两次。使用镊子，将过滤器转移到准备好的玻璃片上，将外植体朝上。装载约50-100μL的嵌入介质。盖上60毫米长的1.5盖玻片。允许嵌入介质溶液在黑暗中室温固化2小时。继续成像或存放幻灯片，包裹在箔中，在4°C直到准备好成像。 </li><li>具有宽视场荧光显微镜7的图像/ sup&gt;耦合到汞灯，并使用用于蓝色激发的反射镜单元（激发带通，460-495nm;双色镜，505nm;发射带通，510-550nm）和20X Plan-Apochromat透镜，0.75数值光圈。 </li></ol>

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</ol>

作者没有什么可以披露的。

该手稿描述了一种从小鼠胚胎中分离出发育中的甲基化成核细胞并培养器官基因的方法。这种方法是由Grobstein 8和Saxén9,10开发的一种标准技术，并被许多其他11,12修改和修改。该方法的成功主要取决于解剖的持续时间，随着剥离时间的延长，外植体存活和诱导电位降低。在清洁周围组织的肾脏基础时，还要注意不要损伤间质。间质间质的损伤通常是外植体生长?...

The mammalian kidney is derived from two primordial structures with mesodermal origin: the tubular epithelial ureteric bud and the metanephric mesenchyme. During nephrogenesis, the ureteric bud invades the metanephric mesenchyme and branches to form the collecting system. The metanephric mesenchyme gives rise to the epithelial elements of the nephrons. These processes occur in a precisely timed and spatially coordinated manner and are initiated by reciprocal inductive mechanisms. Both tissue components communicate and affect the other's cell morphogenesis. 
In the 1920s, it was Boyden who performed the in vivo obstruction of the mesonephric duct in chicken, providing the first indication of inductive interactions as separated nephric blastema fail to differentiate1. At about the same time, the first successful attempts to culture chicken nephric rudiments in a hanging drop were published. Subsequently, the organ culture was developed to study tissue interactions in mammalian organogenesis. In the 1950s, Grobstein developed a technique in which metanephric rudiments could be cultured on a filter. This technique was modified by Sax&#233;n, who placed the filter on a Trowell-type screen in a culture dish1. Over the years, many modifications and applications for organ culture have emerged. The method described here is based on Sax&#233;n's technique but is simplified, as the filters float free on the medium and the diameter of the culture well only slightly exceeds the diameter of the filter, limiting unwanted movement of the filter.
Whole-organ culture is a classical, cheap, and simple but powerful tool to investigate cellular processes and intercellular communication during organogenesis. Organ culture allows for treatment with biological agents, such as growth factors, antibodies, antisense oligonucleotides, viruses, and peptides, as well as with pharmaceutical compounds and other chemicals. Also, gene function may be studied using explants derived from genetically modified mice or using inducible gene inactivation technology, such as the Cre-loxP system. This allows for the study of genetic mutations that cause embryonic lethality prior to the development of the kidney. Organ culture can also be combined with fluorescent tagging for gene function or lineage tracing and modern imaging techniques, which enable real-time monitoring of cell behavior2.
In the specific example provided here, the effect of EphrinB2-activated Eph-receptor signaling on the branching morphology of the ureteric bud was investigated. The morphology of the EphA4/EphB2 double-knockout mice suggested several severe defects in kidney development, which were detectable as early as embryonic day 11 (E11) and involved the ureteric bud, the ureter, and the common nephric duct3. Signaling via Eph receptors requires the clustering of the ligand-receptor dimer4. To over-activate Eph signaling, the kidney rudiments from E11.5 mouse embryos were cultured in the presence of clustered recombinant EphrinB2-Fc. EphrinB2 is a known ligand for the EphA4 receptor, which is expressed in the ureteric bud tips3.

<table><tbody><tr><td>DMEM/F-12</td><td>Thermo Fisher Scientific</td><td>21331020</td><td></td></tr><tr><td>Penicillin-Streptomycin (10,000 U/mL)</td><td>Thermo Fisher Scientific</td><td>15140148</td><td></td></tr><tr><td>GlutaMAX Supplement</td><td>Thermo Fisher Scientific</td><td>35050061</td><td></td></tr><tr><td>DPBS, calcium, magnesium</td><td>Thermo Fisher Scientific</td><td>14040117</td><td>use for dissection</td></tr><tr><td>holo-Transferrin human</td><td>Sigma-Aldrich</td><td>T0665</td><td></td></tr><tr><td>Insulin-Transferrin-Selenium (ITS -G) (100x)</td><td>Thermo Fisher Scientific</td><td>41400045</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>158127</td><td></td></tr><tr><td>Amphotericin B solution</td><td>Sigma-Aldrich</td><td>A2942</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>X100</td><td></td></tr><tr><td>Sodium azide</td><td>Sigma-Aldrich</td><td>S8032</td><td></td></tr><tr><td>Thimerosal</td><td>Sigma-Aldrich</td><td>T5125</td><td></td></tr><tr><td>Propyl gallate</td><td>Sigma-Aldrich</td><td>2370</td><td></td></tr><tr><td>Mowiol 4-88</td><td>Sigma-Aldrich</td><td>81381</td><td></td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G5516</td><td></td></tr><tr><td>Biotinylated Dolichorus Biflorus Agglutinin</td><td>Vector Laboratories</td><td>B-1035</td><td></td></tr><tr><td>Alexa488 conjugated Streptavidin</td><td>Jackson Immuno Research</td><td>016-540-084</td><td></td></tr><tr><td>Recombinant Mouse Ephrin-B2 Fc Chimera Protein, CF</td><td>R&amp;amp;D Systems</td><td>496-EB</td><td></td></tr><tr><td>Recombinant Human IgG1 Fc, CF</td><td>R&amp;amp;D Systems</td><td>110-HG-100</td><td></td></tr><tr><td>Goat Anti-Human IgG Fc Antibody</td><td>R&amp;amp;D Systems</td><td>G-102-C</td><td></td></tr><tr><td>Phosphate buffered saline tablets</td><td>Sigma-Aldrich</td><td>P4417</td><td>use for fixation and immunostaining</td></tr><tr><td>Dumont #5, biologie&lt;br/&gt; tips, INOX, 11 cm</td><td>agnthos.se</td><td>0208-5-PS</td><td>2 pairs of forceps are needed</td></tr><tr><td>Iris scissors, straight, 12 cm</td><td>agnthos.se</td><td>03-320-120</td><td></td></tr><tr><td>Dressing Forceps,&lt;br/&gt; straight, delicate, 13 cm</td><td>agnthos.se</td><td>08-032-130</td><td></td></tr><tr><td>Petri dishes Nunclo Delta treated</td><td>Thermo Fisher Scientific</td><td>150679</td><td></td></tr><tr><td>TMTP01300 Isopore Membrane Filter, polycarbonate, Hydrophilic, 5.0 &amp;micro;m, 13 mm, white, plain</td><td>MerckMillipore</td><td>TMTP01300</td><td></td></tr><tr><td>Nunclon Multidishes&lt;br/&gt; 4 wells, flat bottom</td><td>Sigma-Aldrich</td><td>D6789-1CS</td><td></td></tr><tr><td>Microscope cover glass 24 x 50 mm thickn. No.1.5H 0.17+/-0.005 mm</td><td>nordicbiolabs</td><td>107222</td><td></td></tr><tr><td>Cover glasses No.1.5, 18 mm x 18 mm</td><td>nordicbiolabs</td><td>102032</td><td></td></tr><tr><td>Slides ~76 x 26 x 1, 1/2-w. ground plain</td><td>nordicbiolabs</td><td>1030418</td><td></td></tr><tr><td>VWR Razor Blades</td><td>VWR</td><td>55411-055</td><td></td></tr><tr><td>50 mL centrifuge tubes</td><td>Sigma-Aldrich</td><td>CLS430828</td><td></td></tr><tr><td>15 mL centrifuge tubes</td><td>Sigma-Aldrich</td><td>CLS430055</td><td></td></tr><tr><td>Whatman prepleated qualitative filter paper, Grade 113V, creped</td><td>Sigma-Aldrich</td><td>WHA1213125</td><td></td></tr><tr><td>Fixed stage research mircoscope</td><td>Olympus</td><td>BX61WI</td><td></td></tr><tr><td>Black 6 inbred mice, male, C57BL/6NTac</td><td>Taconic</td><td>B6-M</td><td></td></tr><tr><td>Black 6 inbred mice,female, C57BL/6NTac</td><td>Taconic</td><td>B6-F</td><td></td></tr><tr><td>Greenough Stereo Microscope</td><td>Leica</td><td>Leica S6 E</td><td></td></tr></tbody></table>

dissection and culture of mouse embryonic kidney

该方案的目的是描述一种用于解剖，分离和培养小鼠睾丸的初步方法。 在哺乳动物肾脏发育期间，两个祖细胞组织，输尿管芽和间质间质介导并相互诱导细胞机制，最终形成肾脏的收集系统和肾单位。由于哺乳动物胚胎生长在宫内，因此观察者无法接近，已经开发出器官培养。通过这种方法，可以研究肾脏器官发生期间的上皮 - 间质相互作用和细胞行为。此外，可以研究先天性肾脏和泌尿生殖道畸形的起源。经仔细解剖后，将met ric r are are are are onto onto that that on。。。。。。。。。。。。。。。。。。。。。。。但是，必须意识到条件是人造并可能影响组织中的新陈代谢。此外，由于外植体中存在的细胞外基质和基底膜，测试物质的渗透可能受到限制。 器官文化的一个主要优点是实验者可以直接进入器官。该技术便宜，简单，并且可以进行大量的修改，例如添加生物活性物质，遗传变异体的研究以及先进成像技术的应用。

肾脏肾脏成纤维细胞源于怀孕的黑-65近交小鼠E11.5，并进行培养。 3天后，输尿管芽分支达5次，最终导致T型输尿管芽的分枝。拍摄每个外植体，并对片段和端点的数量进行量化，以确定分枝代数并计算每个分枝的端点数（ 图1 ）。 ImageJ（ rd代;只有8％的处理的外植体达到第 4代，而对照外植体的比例为35％）（ 图1b ）。因此，每个...

Watch this Scientific Journal Video about Dissection and Culture of Mouse Embryonic Kidney at JoVE.com

Dissection and Culture of Mouse Embryonic Kidney

该方案描述了一种从小鼠胚胎中分离和培养元肾小球的方法。

小鼠胚胎肾的解剖与培养

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney ...

dissection-and-culture-of-mouse-embryonic-kidney

Research

JoVE Journal

Developmental Biology

12.0K 次观看.  Uppsala University. 该方案描述了一种从小鼠胚胎中分离和培养元肾小球的方法。 

视频: Dissection and Culture of Mouse Embryonic Kidney

Neural Tube Closure in Mouse Whole Embryo Culture

Genetic mouse models are an important tool in the study of mammalian neural tube closure (Gray &amp; Ross, 2009; Ross, 2010). However, the study of mouse embryos in utero is limited by our inability to directly pharmacologically manipulate the embryos in isolation from the effects of maternal metabolism on the reagent of interest. Whether using a small molecule, recombinant protein, or siRNA, delivery of these substances to the mother, through the diet or by injection will subject these unstable compounds to a variety of bodily defenses that could prevent them from reaching the embryo. Investigations in cultures of whole embryos can be used to separate maternal from intrinsic fetal effects on development.
Here, we present a method for culturing mouse embryos using highly enriched media in a roller incubator apparatus that allows for normal neural tube closure after dissection (Crockett, 1990). Once in culture, embryos can be manipulated using conventional in vitro techniques that would not otherwise be possible if the embryos were still in utero. Embryo siblings can be collected at various time points to study different aspects of neurulation, occurring from E7-7.5 (neural plate formation, just prior to the initiation of neurulation) to E9.5-10 (at the conclusion of cranial fold and caudal neuropore closure, Kaufman, 1992). In this protocol, we demonstrate our method for dissecting embryos at timepoints that are optimal for the study of cranial neurulation. Embryos will be dissected at E8.5 (approx. 10-12 somities), after the initiation of neural tube closure but prior to embryo turning and cranial neural fold closure, and maintained in culture till E10 (26-28 somities), when cranial neurulation should be complete.

A method allowing for direct pharmacological manipulation of mouse embryos during neurulation that bypasses maternal metabolism is described. The technique can be adapted to study different aspects of neurulation by varying the time point and pharmacological agent.

在小鼠全胚胎培养的神经管封闭

Genetic mouse models are an important tool in the study of mammalian neural tube closure (Gray &amp; Ross, 2009; Ross, 2010).  However, the study of mouse ...

科研

神经科学

Organ Culture and Whole Mount Immunofluorescence Staining of Mouse Wolffian Ducts

Tubal morphogenesis is a fundamental requirement for the development of most mammalian organs, including the male reproductive system. The epididymis, an integral part of the male reproductive tract, is responsible for sperm storage, maturation, and transport. The adult epididymis is a highly coiled tube that develops from a simple and straight embryonic precursor known as Wolffian duct (WD). Proper coiling of the epididymis is essential for male fertility, as sperm in the testis are unable to fertilize an oocyte. However, the mechanism responsible for epididymal development and coiling remains unclear, partially due to the lack of whole organ culture and imaging methods. In this study, we describe an in vitro culture system and whole mount immunofluorescence protocol to better visualize the process of WD coiling and development, which may also be applied to study other tubular organs.

We present a method for isolation and culture of the mouse Wolffian duct (WD). We also demonstrate a detailed procedure for whole mount immunostaining of cultured/freshly isolated WDs with fluorescently tagged antibodies. Together, these techniques enable the study of WD development, coiling, and differentiation.

器官培养和鼠标沃尔弗导管的全山免疫染色

Tubal morphogenesis is a fundamental requirement for the development of most mammalian organs, including the male reproductive system. The epididymis, an ...

发育生物学

Optimization of Renal Organoid and Organotypic Culture for Vascularization, Extended Development, and Improved Microscopy Imaging

Embryonic kidney organotypic cultures, and especially pluripotent stem cell-derived kidney organoids, are excellent tools for following developmental processes and modelling kidney disease. However, the models are limited by a lack of vascularization and functionality. To address this, an improved protocol for the method of xenografting cells and tissues to the chorioallantoic membrane (CAM) of an avian embryo to gain vascularization and restoration of blood flow was developed. The grafts are overlaid with custom-made minireservoirs that fix the samples to the CAM and supply them with culture medium that protects the grafts from drying. The improved culture method allows xenografts to grow for up to 9 days. The manuscript also describes how to provide optimal conditions for long-term confocal imaging of renal organoids and organotypic cultures using the previously published Fixed Z-Direction (FiZD) method. This method gently compresses an embryonic organ or organoid between a glass coverslip and membrane in a large amount of medium and provides excellent conditions for imaging for up to 12 days. Together, these methods allow vascularization and blood flow to renal organoids and organotypic kidney cultures with improved confocal imaging. The methods described here are highly beneficial for studying fundamental and applied functions of kidneys ex vivo. Both methods are applicable to various types of tissues and organoids.

This work describes two methods for studying organ development, an improved xenotransplantation setup on chorioallantoic membrane (CAM) from avian embryos that allows for vascularization of cultured embryonic organs and organoids and a novel fixed z-direction organ culture method with modified experimental conditions that allows for high-resolution time-lapse confocal imaging.

血管化、扩展发育和改进显微镜成像的肾器官和器官培养的优化

Embryonic kidney organotypic cultures, and especially pluripotent stem cell-derived kidney organoids, are excellent tools for following developmental ...

Mouse Embryonic Lung Culture, A System to Evaluate the Molecular Mechanisms of Branching

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of the lung endoderm with its surrounding mesenchyme and mesothelium. Lung mesenchyme has been shown to be the source of inductive signals for lung branching morphogenesis. Epithelial-mesenchymal-mesothelial interactions are also critical to embryonic lung morphogenesis. Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system (in the absence of maternal influence and blood flow). More importantly this technique can be readily done in a serumless, chemically defined culture media. Gain and loss of function can be achieved using expressed proteins, recombinant viral vectors and/or analysis of transgenic mouse strains, antisense RNA, as well as RNA interference gene knockdown.

Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system.

小鼠胚胎的龙文化，一个系统来评价分枝的分子机制

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of ...

生物学

Microdissection of Primary Renal Tissue Segments and Incorporation with Novel Scaffold-free Construct Technology

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only one-fourth of these patients achieving transplantation, there is a dire need for alternatives for those with failing organs. In order to decrease the harmful consequences of dialysis along with the overall healthcare costs it incurs, active investigation is ongoing in search of alternative solutions to organ transplantation. Implantable tissue-engineered renal cellular constructs are one such feasible approach to replacing lost renal functionality. Here, described for the first time, is the microdissection of murine kidneys for isolation of living corticomedullary renal segments. These segments are capable of rapid incorporation within scaffold-free endothelial-fibroblast constructs which may enable rapid connection with host vasculature once implanted. Adult mouse kidneys were procured from living donors, followed by stereoscope microdissection to obtain renal segments 200 - 300 &#181;m in diameter. Multiple renal constructs were fabricated using primary renal segments harvested from only one kidney. This method demonstrates a procedure which could salvage functional renal tissue from organs that would otherwise be discarded.

Tissue-engineered renal constructs provide a solution for the organ shortage and deleterious effects of dialysis. Here, we describe a protocol to micro dissect murine kidneys for isolation of cortico-medullary segments. These segments are implanted into scaffold-free cellular constructs, forming renal organoids.

原发性肾组织段的显微解剖与新型无支架结构技术的结合

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only ...

生物工程

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and to gain understanding of the origins of congenital kidney disease. More recently, the possibility of steering na&iuml;ve embryonic stem cells toward nephrogenic fates has been explored in the emerging field of regenerative medicine. Genetic studies in the mouse have identified several pathways required for kidney development, and a global catalog of gene transcription in the organ has recently been generated <a href="http://www.gudmap.org/">http://www.gudmap.org/</a>, providing numerous candidate regulators of essential developmental functions. Organogenesis of the rodent kidney can be studied in organ culture, and many reports have used this approach to analyze outcomes of either applying candidate proteins or knocking down the expression of candidate genes using siRNA or morpholinos. However, the applicability of organ culture to the study of signaling that regulates stem/progenitor cell differentiation versus renewal in the developing kidney is limited as cultured organs contain a compact extracellular matrix limiting diffusion of macromolecules and virus particles. To study the cell signaling events that influence the stem/progenitor cell niche in the kidney we have developed a primary cell system that establishes the nephrogenic zone or progenitor cell niche of the developing kidney ex vivo in isolation from the epithelial inducer of differentiation. Using limited enzymatic digestion, nephrogenic zone cells can be selectively liberated from developing kidneys at E17.5. Following filtration, these cells can be cultured as an irregular monolayer using optimized conditions. Marker gene analysis demonstrates that these cultures contain a distribution of cell types characteristic of the nephrogenic zone in vivo, and that they maintain appropriate marker gene expression during the culture period. These cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which greatly facilitates the study of candidate stem/progenitor cell regulator effects. Basic cell biological parameters such as proliferation and cell death as well as changes in expression of molecular markers characteristic of nephron stem/progenitor cells in vivo can be successfully used as experimental outcomes. Ongoing work in our laboratory using this novel primary cell technique aims to uncover basic mechanisms governing the regulation of self-renewal versus differentiation in nephron stem/progenitor cells.

In this report we describe a method for the isolation and culture of the progenitor cell niche from the embryonic mouse kidney that can be used to study signaling pathways regulating stem/progenitor cells of the developing kidney. These cultured cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which allows efficient manipulation of candidate pathways.

从小鼠胚胎肾肾区细胞的分离和培养

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

Isolation and Culture of Embryonic Mouse Neural Stem Cells

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and expansion of NSCs provide a suitable source of cells for neuroscientists to study the function of neurons and glial cells along with their interactions. There are several reported techniques for the isolation of neural stem cells from adult or embryo mammalian brains. During the microsurgical operation to isolate NSCs from different regions of the embryonic CNS, it is very important to reduce the damage to the brain cells to obtain the highest ratio of live and expandable stem cells. A possible technique for stress reduction during isolation of these cells from the mouse embryo brain is the reduction of surgical time. Here, we demonstrate a developed technique for rapid isolation of these cells from the E13 mouse embryo ganglionic eminence. Surgical procedures include harvesting E13 mouse embryos from the uterus, cutting the frontal fontanelle of the embryo with a bent needle tip, extracting the brain from the skull, microdissection of the isolated brain to harvest the ganglionic eminence, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in suspension culture to generate neurospheres.

Here, we introduce a novel microsurgical technique for the isolation of neural stem cells from the E13 mouse embryo ganglionic eminence.

胚胎小鼠神经干细胞的分离与培养

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and ...

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O2 / 5% CO2 in a rolling bottle culture apparatus at 37 &#176;&#8203;C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.

Serum utilized in embryo cultures contains unknown components that can affect the outcome of experiments especially in studies involving signaling interactions. Here we utilized a serum-free oxygenated culture system and show that mid-gestation mouse embryos cultured for 16-40 hr&#160;exhibit morphological development comparable to embryos developing in utero.

鼠胚体外发育无血清全胚胎培养系统

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in ...

Glomerular Outgrowth as an Ex Vivo Assay to Analyze Pathways Involved in Parietal Epithelial Cell Activation

Parietal epithelial cell (PEC) activation is one of the key factors involved in the development and progression of glomerulosclerosis. Inhibition of pathways involved in parietal epithelial cell activation could therefore be a tool to attenuate the progression of glomerular diseases. This article describes a method to culture and analyze parietal epithelial cell outgrowth of encapsulated glomeruli isolated from mouse kidney. After dissecting isolated mouse kidneys, the tissue is minced, and glomeruli are isolated by sieving. Encapsulated glomeruli are collected, and single glomeruli are cultured for 6 days to obtain glomerular outgrowth of parietal epithelial cells. During this period, parietal epithelial cell proliferation and migration can be analyzed by determining the cell number or the surface area of outgrowing cells. This assay can therefore be used as a tool to study the effects of an altered gene expression in transgenic- or knockout-mice or the effects of culture conditions on parietal epithelial cell growth characteristics and signaling. Using this method, important pathways involved in the process of parietal epithelial cell activation and consequently in glomerulosclerosis can be studied.

This article describes a method for culturing and analyzing glomerular parietal epithelial cell outgrowths of encapsulated glomeruli isolated from mouse kidney. This method can be used to study pathways involved in parietal epithelial cell proliferation and migration.

球状外生长作为前维沃分析分析涉及的路径的表皮上皮细胞激活

Parietal epithelial cell (PEC) activation is one of the key factors involved in the development and progression of glomerulosclerosis. Inhibition of ...

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to understand the pathophysiology of ocular diseases because of their similarities with human anatomy and physiology. Alterations in the retinal pigment epithelium (RPE), including changes in morphology and function, are common features shared by many ocular disorders. However, successful isolation and culture of primary mouse RPE cells is very challenging. This paper is an updated audiovisual version of the protocol previously published by Fernandez-Godino et al. in 2016 to efficiently isolate and culture primary mouse RPE cells. This method is highly reproducible and results in robust cultures of highly polarized and pigmented RPE monolayers that can be maintained for several weeks on Transwells. This model opens new avenues for the study of the molecular and cellular mechanisms underlying eye diseases. Moreover, it provides a platform to test therapeutic approaches that can be used to treat important eye diseases with unmet medical needs, including inherited retinal disorders and macular degenerations.

This protocol, which was originally reported by Fernandez-Godino et al. in 20161, describes a method to efficiently isolate and culture mouse RPE cells, which form a functional and polarized RPE monolayer within one week on Transwell plates. The procedure takes approximately 3 hours.

原发性小鼠视网膜色素上皮细胞的有效解剖与培养

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to ...